Biofilms are complex bacterial communities attached to a surface. The formation of biofilms has a profound impact on human health and industrial processes and has been recognized as an important, but poorly studied aspect of the bacterial life cycle, Biofilm development includes a transition from individual, planktonic (free-swimming) bacteria to a mode of life attached to a surface as part of a multi-cellular community, and the subsequent return to a planktonic existence. One of the most widely recognized properties of bacteria growing in a biofilm is their increased resistance to antimicrobial agents. Despite increasing interest in understanding how resistance develops, little is known about the molecular mechanisms that drive biofilms cells towards this highly resistant state. Our evidence supports the central hypothesis of this proposal: antimicrobial resistance of biofilm-grown cells requires distinct genetic elements. Herein, we propose a series of complimentary approaches to elucidate molecular mechanisms underlying biofilm-specific antibiotic resistance using three model antibiotics. While part of the proposed work will continue our efforts to study known biofilm-specific functions in P. aeruginosa (Specific Aim I), work described in Specific Aims II and III will begin in depth analysis of two newly discovered genetic loci required for resistance to antibiotics in a biofilm. The multiple experimental approaches outlined in this proposal should lead to a better understanding of the mechanism(s) involved in development of biofilm-specific antibiotic resistance, and potentially to new therapeutic strategies for modulating these properties. Specific Aim I. Studies of known genes in biofilm-specific antibiotic resistance. Specific Aim II. Determine the role of a glucan synthetase enzyme in the development of biofilm-specific antibiotic resistance. Specific Aim III. Studies of the role of a putative new efflux pump in biofilm-specific antibiotic resistance.